Targeted therapy for small cell lung cancer

ABSTRACT

Methods are provided for treatment of lung cancers, particularly small cell lung cancer with targeted therapy, which optionally includes an agent that selectively blocks CD47 binding to SIRPα.

CROSS REFERENCE

This application is a Divisional and claims the benefit of U.S.application Ser. No. 15/107,852, filed Jun. 23, 2016, which claims thebenefit of 371 Application No. PCT/US2015/010650, filed Jan. 8, 2015,which claims the benefit of U.S. Provisional Application No. 61/925,143,filed Jan. 8, 2014, which are incorporated herein in their entirety forall purposes.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith a text file,(S13-499_STAN-1089DIV_Sequence_listinglist.txt), created on (Nov. 18,2020) and having a size of (2 KB) The contents of the text file areincorporated by reference herein in their entirety.

BACKGROUND

Targeted therapies, such as antibodies and specific ligands have proveneffective at fighting cancer, especially in cases where conventionaltherapy fails. Even more encouraging is that antibodies for cancergenerally operate in a distinct mechanism from traditional chemotherapyor radiotherapy, so they can often be combined with traditionaltherapies to generate an additive or synergistic effect.

Antibodies can achieve their therapeutic effect through variousmechanisms. They can have direct effects in producing apoptosis orprogrammed cell death. They can block growth factor receptors,effectively arresting proliferation of tumor cells. In cells thatexpress monoclonal antibodies, they can bring about anti-idiotypeantibody formation. Indirect effects include recruiting cells that havecytotoxicity, such as monocytes and macrophages. This type ofantibody-mediated cell kill is called antibody-dependent cell mediatedcytotoxicity (ADCC). Monoclonal antibodies also bind complement, leadingto direct cell toxicity, known as complement dependent cytotoxicity(CDC).

CD47 is a valuable target for anticancer therapy due to its function asan inhibitor of macrophage phagocytosis as well as its broad expressionon a variety of human neoplasms. By binding to signal-regulatory proteinα (SIRPα), a receptor expressed on the surface of macrophages, CD47 isable to transduce inhibitory signals that prevent phagocytosis. Blockingthe interaction between CD47 and SIRPα with antibodies not onlystimulates macrophages to engulf cancer cells in vitro but also exertsrobust anticancer effects in vivo. Other CD47 blocking agents include“next-generation” CD47 antagonists that bind and block human CD47 withextraordinarily high affinity.

By disabling the inhibitory signals transduced by SIRPα, high-affinitySIRPα variants can reduce the threshold for macrophage activation andpromote phagocytic response driven by tumor-specific antibodies. Thedegree to which the anticancer activity of a given therapeutic antibodyis enhanced by CD47 blockade likely depends on multiple factors,including the levels of antigen expression on the surface of malignantcells, the isotype of its heavy chain, and the orientation assumed bythe antibody upon antigen binding, which affects its ability to engageFc receptors on immune effectors. High-affinity SIRPα monomers representtherefore a rapid, safe and effective alternative to several otherapproaches, including drug/toxin conjugation strategies, that have beenpursued in this direction.

Identification of effective targets and combinations of targetedtherapies remain of high interest. The present invention addresses thisneed.

SUMMARY OF THE INVENTION

Methods and compositions are provided for the treatment of lung cancerwith a targeted therapy. In some embodiments, the lung cancer is smallcell lung cancer. In some embodiments, the therapy is targeted at one ormore cell-surface antigens, including CD24, CD166, CD56, CD326, CD298,CD29, CD63, CD9, CD164, CD99, CD46, CD59, CD57, CD165, EpCAM, etc. Insome embodiments the targeted therapy comprises administering to anindividual suffering from lung cancer a therapeutic dose of an antibodythat specifically binds to a cell surface marker selected from CD24,CD166, CD56, CD326, CD298, CD29, CD63, CD9, CD164, CD99, CD46, CD59,CD57, CD165 and EpCAM.

In some embodiments the targeted therapy is combined with a CD47blocking agent. Cancer cells evade macrophage surveillance byupregulation of CD47 expression. Administration of agents that mask theCD47 protein, e.g. antibodies or small molecules that bind to CD47 orSIRPα and prevent interaction between CD47 and SIRPα, are administeredto a patient, which increases the clearance of cancer cells viaphagocytosis. The agent that blocks CD47 is combined with monoclonalantibodies directed against one or more lung cancer cell markers, whichcompositions can be synergistic in enhancing phagocytosis andelimination of cancer cells as compared to the use of single agents.

Specific reagent combinations of interest for therapy include anti-CD47and anti-CD56; anti-CD47 and anti-CD44, anti-CD47 and anti-CD99,anti-CD47 and anti-EpCam. In some such embodiments the anti-CD47 reagentis a high affinity SIRPα polypeptide, which can be provided in the formof a monomer or a multimer, e.g. as a fusion protein with an IgG Fcpolypeptide.

In other embodiments, the therapy provides for a multispecific antibodythat targets CD47 and a second cancer cell marker, includingmultispecific antibodies that target CD47 and CD56; CD47 and CD44, CD47and EpCam, etc. Compositions of such multispecific antibodies are alsoprovided, where the multispecific antibody is desirably human orhumanized; and may be modified to extend the blood half-life, e.g. bypegylation, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: CD47-blocking therapies stimulate macrophage phagocytosis ofsmall cell lung cancer. Human monocytes from anonymous blood donors werepurified by magnetic activated cell sorting (MACS) using CD14+selection. Monocytes were cultured in the presence of 10% AB human serumfor one week, at which point the exhibited morphological changescharacteristic of differentiation to macrophages. Macrophages wereco-cultured with primary human small cell lung cancer cells (SCLC sample“H29”) labeled with a green fluorescent dye. Cells were treated witheither a vehicle control (phosphate buffered saling, PBS), anti-CD56antibody (clone MEM-188), or humanized anti-CD47 antibody (clone5F9-G4). Phagocytosis was evaluated by high-throughput flow cytometry asthe percentage of macrophages that had engulfed green fluorescent SCLCcells. Treatment with anti-CD47 antibody was able to induce elevatedlevels of phagocytosis as a single agent.

FIG. 2: CD47-blockade produces a therapeutic response against small celllung cancer cells in vivo using mouse xenotransplantation models.Primary small cell lung cancer cells (sample H29) were engrafted intoimmunodeficient NSG mice. After approximately three weeks of growth,mice were randomized into two treatment cohorts. The first cohort wastreated with a vehicle control (phosphate buffered saline, PBS; red),and the second cohort was treated with daily injections of 250 μganti-CD47 antibody (clone 5F9-G4, blue). Tumor growth was monitored overtime. Each point represents a tumor growing in an individual mouse.Black bars represent median tumor volume. Left Tumor volume measurementsover entire time course of study. Right Tumor volume measurements on day89 of study. Note logarithmic scale.

FIG. 3: Novel therapeutic targets highly expressed on the surface ofsmall cell lung cancer cells. Primary human small cell lung cancer cells(sample H29) were subjected to comprehensive flow cytometricimmunophenotyping using a LEGENDScreen assay (Biolegend). Surfaceantigens were ranked based on their geometric mean fluorescenceintensity (Geo. MFI). These antigens are therapeutic targets forantibodies in combination with CD47-blocking agents.

FIG. 4: The ability of SCLC-targeting antibodies to induce macrophagephagocytosis can be enhanced by combination with CD47-blockingtherapies. Two human small cell lung cancer cell lines (H82 and H69)were labeled with a green fluorescent dye, and then were co-culturedwith primary NSG mouse macrophages in the presence of the indicatedantibodies either in combination with vehicle control (phosphatebuffered saline, PBS; gray) or with high-affinity SIRPalpha variant CV1monomer (black). Phagocytosis was evaluated by high-throughput flowcytometry as the percentage of macrophages that had engulfed greenfluorescent SCLC cells. Anti-CD47 reagents 5F9-G4 and CV1-G4 were nottested in combination with CV1 monomer due to direct competition.

FIG. 5. CD47-blockade induces macrophage phagocytosis of SCLC cells invitro. (A) Expression of CD47 on the surface of a panel of human SCLCcell lines as evaluated by flow cytometry. Black dotted line representsunstained NCI-H82 cells. (B) Expression of CD47 on the surface of theprimary human SCLC sample H29. (C) Diagram depicting in vitrophagocytosis assays using human macrophages and fluorescent tumor cells.(D) Representative flow cytometry plots of phagocytosis assays performedwith human macrophages and calcein AM-labeled SCLC cells. (E)Representative images of cell populations after fluorescence activatedcell sorting. The sorted double-positive population containedmacrophages with engulfed tumor cells. Scale bar represents 20 μm. (F)Summary of phagocytosis assays using human macrophages and calceinAM-labeled SCLC cells as analyzed by flow cytometry. SCLC cells weretreated with vehicle control (PBS) or anti-CD47 antibodies (cloneHu5F9-G4). The percentage of calcein AM+ macrophages was normalized tothe maximal response by each macrophage donor. (G) Phagocytosis ofprimary H29 SCLC cells by human macrophages after treatment with vehiclecontrol (PBS) or anti-CD47 antibodies (clone Hu5F9-G4). (F-G)Phagocytosis assays were performed with macrophages derived from fourindependent blood donors. Data represent mean±SD. ns, not significant;**P<0.01; ****P<0.0001 for the indicated comparisons by two-way analysisof variance with Sidak correction (F) or two-tailed t test (G).

FIG. 6. CD47-blocking antibodies inhibit growth of human SCLC tumors invivo. (A) Growth of NCI-H82 cells in the subcutaneously tissue of NSGmice. Mice were randomized into groups treated with vehicle control(PBS) or anti-CD47 antibodies (clone Hu5F9-G4). Growth was evaluated bytumor volume measurements. Seven to eight mice were treated per cohort,and each point represents tumor volume of independent animals. (B)Growth of GFP-luciferase+ patient-derived xenograft H29 tumors in thesubcutaneous tissue of NSG mice as evaluated by bioluminescence imaging.Mice were randomized into groups treated with vehicle control (PBS) oranti-CD47 antibodies (clone Hu5F9-G4). (C) Representativebioluminescence images of H29 tumors on day 85 post-engraftment. (D)Growth of H29 tumors as evaluated by tumor volume measurements. (E)Survival of mice bearing patient-derived xenograft H29 tumors that weretreated with the indicated therapies. P=0.0004 by Mantel-Cox test. A-EBlack arrows indicate the start of treatment. Points indicatemeasurements from independent animals, bars indicate median values.Cohorts consisted of a minimum of 7-8 mice. Measurements at each timepoint are staggered for clarity. ns, not significant; *P<0.05; **P<0.01;***P<0.001 for the indicated comparisons by Mann-Whitney test.

FIG. 7. MCP-3 is a serum biomarker that predicts response toCD47-blocking therapies. (A) Untreated NSG mice (No tumor) or NSG micebearing subcutaneous NCI-H82 cells were injected with a single dose ofanti-CD47 antibodies (clone Hu5F9-G4). Serum samples were collectedpre-treatment or 24 hours post-treatment. MCP-3 levels were measured byLuminex multiplex array. (B) MCP-3 levels in mice bearingpatient-derived xenograft H29 tumors were evaluated as in A. Pointsrepresent measurements from individual mice, bars represent mean±SD.Five mice were evaluated per condition. ns=not significant; ****P<0.0001by two-way analysis of variance with Sidak correction.

FIG. 8. Comprehensive FACS-based antibody screening identifies new andestablished therapeutic targets on SCLC. Antigen expression on thesurface of four SCLC cell lines and primary patient sample H29 wasassessed using LEGENDScreen Human Cell Screening Kits (BioLegend), acollection of 332 antibodies targeting cell surface antigens. Antibodybinding was detected by fluorescence-activated cell sorting (FACS)analysis. (A) Histogram depicting geometric mean fluorescence intensity(MFI) of all antibodies screened for SCLC surface binding. Datarepresent median values for each antibody across all five SCLC samples.Data were fit to Gaussian distribution (black curve), and negativeantigens (gray) were defined by median MFI less than two standarddeviations above the mean. Low antigens (red) defined as MFI less thanone order of magnitude above the negative threshold. High antigens(blue) defined as one order of magnitude greater than negativethreshold. (B) Ranked list of the 39 antigens identified as ‘high’ basedon median MFI across all five SCLC samples.

FIG. 9. High-affinity SIRPα variants enhance macrophage phagocytosis ofSCLC in response to tumor-binding antibodies. Phagocytosis of NCI-H82cells (A) and NCI-H524 cells (B) in response to tumor-binding antibodiesalone (red) or in combination with the high-affinity SIRPα variant CV1monomer (blue). Points represent measurements from individual donors,bars represent median values. Three clones of anti-CD56 (NCAM)antibodies were tested, as well as antibodies to CD24, CD29, CD99, andCD47 (clone Hu5F9-G4). (C) Phagocytosis of NCI-H82 SCLC cells inresponse to varying concentrations of the anti-CD56 antibodylorvotuzumab alone (red) or in combination with the high-affinity SIRPαvariant CV1 monomer (blue). Data represent mean±SD. (A-C) Phagocytosisassays were performed with human macrophages derived from a minimum offour independent blood donors. Measurements were normalized to themaximal response by each macrophage donor. ns, not significant; *P<0.05;**P<0.01; ***P<0.001; ****P<0.0001 for the indicated comparisons bytwo-way analysis of variance with Sidak correction.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Methods and compositions are provided for the treatment of lung cancerwith a therapeutic agent, e.g. an antibody, targeted to a marker of lungcancer, e.g. targeted to one or more cell-surface antigens, includingCD24, CD166, CD56, CD326, CD298, CD29, CD63, CD9, CD164, CD99, CD46,CD59, CD57, CD165, EpCAM, etc. In some embodiments, a combination, e.g.a synergistic combination, of agents is provided, wherein one agent isan anti-CD47 blocking agent, and the second agent is targeted to a lungcancer marker, e.g. CD24, CD166, CD56, CD326, CD298, CD29, CD63, CD9,CD164, CD99, CD46, CD59, CD57, CD165, EpCAM, etc.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Methods recited herein may be carried out in any order of the recitedevents which is logically possible, as well as the recited order ofevents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

Synergistic combination. Synergistic combinations may provide for atherapeutic effect that is comparable to the effectiveness of amonotherapy, i.e. the individual components of the combination, whilereducing adverse side effects, e.g. damage to non-targeted tissues,immune status, and other clinical indicia. Alternatively synergisticcombinations may provide for an improved effectiveness when compared tothe effectiveness of a monotherapy, i.e. the individual components ofthe combination, which effect may be measured by total tumor cellnumber; length of time to relapse; and other indicia of patient health.

Synergistic combinations of the present invention combine an agent thatis targeted to inhibit or block CD47 function; and an agent that istargeted to inhibit or block a second lung cancer cell marker, usually acell surface marker. The combination may be provided with a combinationof agents, e.g. two distinct proteins, each of which is specific for adifferent marker; or may be provided as a multispecific agent, e.g.antibody, that combines specificity for two or more different markers.

Combination Therapy: As used herein, the term “combination therapy”refers to those situations in which a subject is simultaneously exposedto two or more therapeutic regimens (e.g., two or more therapeuticagents). In some embodiments, two or more agents may be administeredsimultaneously; in some embodiments, such agents may be administeredsequentially; in some embodiments, such agents are administered inoverlapping dosing regimens.

Dosage Form: As used herein, the term “dosage form” refers to aphysically discrete unit of an active agent (e.g., a therapeutic ordiagnostic agent) for administration to a subject. Each unit contains apredetermined quantity of active agent. In some embodiments, suchquantity is a unit dosage amount (or a whole fraction thereof)appropriate for administration in accordance with a dosing regimen thathas been determined to correlate with a desired or beneficial outcomewhen administered to a relevant population (i.e., with a therapeuticdosing regimen). Those of ordinary skill in the art appreciate that thetotal amount of a therapeutic composition or agent administered to aparticular subject is determined by one or more attending physicians andmay involve administration of multiple dosage forms.

Dosing Regimen: As used herein, the term “dosing regimen” refers to aset of unit doses (typically more than one) that are administeredindividually to a subject, typically separated by periods of time. Insome embodiments, a given therapeutic agent has a recommended dosingregimen, which may involve one or more doses. In some embodiments, adosing regimen comprises a plurality of doses each of which areseparated from one another by a time period of the same length; in someembodiments, a dosing regimen comprises a plurality of doses and atleast two different time periods separating individual doses. In someembodiments, all doses within a dosing regimen are of the same unit doseamount. In some embodiments, different doses within a dosing regimen areof different amounts. In some embodiments, a dosing regimen comprises afirst dose in a first dose amount, followed by one or more additionaldoses in a second dose amount different from the first dose amount. Insome embodiments, a dosing regimen comprises a first dose in a firstdose amount, followed by one or more additional doses in a second doseamount same as the first dose amount. In some embodiments, a dosingregimen is correlated with a desired or beneficial outcome whenadministered across a relevant population (i.e., is a therapeutic dosingregimen).

CD47 polypeptides. The three transcript variants of human CD 47 (variant1, NM 001777; variant 2, NM 198793; and variant 3, NM 001025079) encodethree isoforms of CD47 polypeptide. CD47 isoform 1 (NP 001768), thelongest of the three isoforms, is 323 amino acids long. CD47 isoform 2(NP 942088) is 305 amino acid long. CD47 isoform 3 is 312 amino acidslong. The three isoforms are identical in sequence in the first 303amino acids. Amino acids 1-8 comprise the signal sequence, amino acids9-142 comprise the CD47 immunoglobulin like domain, which is the solublefragment, and amino acids 143-300 is the transmembrane domain.

A “functional derivative” of a native sequence polypeptide is a compoundhaving a qualitative biological property in common with a nativesequence polypeptide. “Functional derivatives” include, but are notlimited to, fragments of a native sequence and derivatives of a nativesequence polypeptide and its fragments, provided that they have abiological activity in common with a corresponding native sequencepolypeptide. The term “derivative” encompasses both amino acid sequencevariants of polypeptide and covalent modifications thereof. Derivativesand fusion of soluble CD47 find use as CD47 mimetic molecules.

The first 142 amino acids of CD47 polypeptide comprise the extracellularregion of CD47 (SEQ ID NO: 1). The three isoforms have identical aminoacid sequence in the extracellular region, and thus any of the isoformsare can be used to generate soluble CD47. “Soluble CD47” is a CD47protein that lacks the transmembrane domain. Soluble CD47 is secretedout of the cell expressing it instead of being localized at the cellsurface.

In vitro assays for CD47 biological activity include, e.g. inhibition ofphagocytosis of porcine cells by human macrophages, binding to SIRP αreceptor, SIRP α tyrosine phosphorylation, etc. An exemplary assay forCD47 biological activity contacts a human macrophage composition in thepresence of a candidate agent. The cells are incubated with thecandidate agent for about 30 minutes and lysed. The cell lysate is mixedwith anti-human SIRP α antibodies to immunoprecipitate SIRP α.Precipitated proteins are resolved by SDS PAGE, then transferred tonitrocellulose and probed with antibodies specific for phosphotyrosine.A candidate agent useful as a CD47 mimetic increases SIRP α tyrosinephosphorylation by at least 10%, or up to 20%, or 50%, or 70% or 80% orup to about 90% compared to the level of phosphorylation observed in theabsence of candidate agent. Another exemplary assay for CD47 biologicalactivity measures phagocytosis of hematopoietic cells by humanmacrophages. A candidate agent useful as a CD47 mimetic results in thedown regulation of phagocytosis by at least about 10%, at least about20%, at least about 50%, at least about 70%, at least about 80%, or upto about 90% compared to level of phagocytosis observed in absence ofcandidate agent.

By “manipulating phagocytosis” is meant an up-regulation or adown-regulation in phagocytosis by at least about 10%, or up to 20%, or50%, or 70% or 80% or up to about 90% compared to level of phagocytosisobserved in absence of intervention. Thus in the context of decreasingphagocytosis of circulating hematopoietic cells, particularly in atransplantation context, manipulating phagocytosis means adown-regulation in phagocytosis by at least about 10%, or up to 20%, or50%, or 70% or 80% or up to about 90% compared to level of phagocytosisobserved in absence of intervention.

Anti-CD47 agent. As used herein, the term “anti-CD47 agent” refers toany agent that reduces the binding of CD47 (e.g., on a target cell) toSIRPα (e.g., on a phagocytic cell). Non-limiting examples of suitableanti-CD47 reagents include SIRPα reagents, including without limitationhigh affinity SIRPα polypeptides, anti-SIRPα antibodies, soluble CD47polypeptides, and anti-CD47 antibodies or antibody fragments. In someembodiments, a suitable anti-CD47 agent (e.g. an anti-CD47 antibody, aSIRPα reagent, etc.) specifically binds CD47 to reduce the binding ofCD47 to SIRPα. In some embodiments, a suitable anti-CD47 agent (e.g., ananti-SIRPα antibody, a soluble CD47 polypeptide, etc.) specificallybinds SIRPα to reduce the binding of CD47 to SIRPα. A suitable anti-CD47agent that binds SIRPα does not activate SIRPα (e.g., in theSIRPα-expressing phagocytic cell).

The efficacy of a suitable anti-CD47 agent can be assessed by assayingthe agent (further described below). In an exemplary assay, target cellsare incubated in the presence or absence of the candidate agent. Anagent for use in the methods of the invention will up-regulatephagocytosis by at least 10% (e.g., at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 100%, at least 120%, at least 140%, at least 160%, atleast 180%, or at least 200%) compared to phagocytosis in the absence ofthe agent. Similarly, an in vitro assay for levels of tyrosinephosphorylation of SIRPα will show a decrease in phosphorylation by atleast 5% (e.g., at least 10%, at least 15%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or 100%) compared to phosphorylation observed in absence ofthe candidate agent.

In some embodiments, the anti-CD47 agent does not activate CD47 uponbinding. When CD47 is activated, a process akin to apoptosis (i.e.,programmed cell death) may occur (Manna and Frazier, Cancer Research,64, 1026-1036, Feb. 1 2004). Thus, in some embodiments, the anti-CD47agent does not directly induce cell death of a CD47-expressing cell byapoptosis.

SIRPα reagent. A SIRPα reagent comprises the portion of SIRPα that issufficient to bind CD47 at a recognizable affinity, which normally liesbetween the signal sequence and the transmembrane domain, or a fragmentthereof that retains the binding activity. A suitable SIRPα reagentreduces (e.g., blocks, prevents, etc.) the interaction between thenative proteins SIRPα and CD47. The SIRPα reagent will usually compriseat least the dl domain of SIRPα. In some embodiments, a SIRPα reagent isa fusion protein, e.g., fused in frame with a second polypeptide. Insome embodiments, the second polypeptide is capable of increasing thesize of the fusion protein, e.g., so that the fusion protein will not becleared from the circulation rapidly. In some embodiments, the secondpolypeptide is part or whole of an immunoglobulin Fc region. The Fcregion aids in phagocytosis by providing an “eat me” signal, whichenhances the block of the “don't eat me” signal provided by the highaffinity SIRPα reagent. In other embodiments, the second polypeptide isany suitable polypeptide that is substantially similar to Fc, e.g.,providing increased size, multimerization domains, and/or additionalbinding or interaction with Ig molecules.

In some embodiments, a subject anti-CD47 agent is a “high affinity SIRPαreagent”, which includes SIRPα-derived polypeptides and analogs thereof.High affinity SIRPα reagents are described in international applicationPCT/US13/21937, which is hereby specifically incorporated by reference.High affinity SIRPα reagents are variants of the native SIRPα protein.In some embodiments, a high affinity SIRPα reagent is soluble, where thepolypeptide lacks the SIRPα transmembrane domain and comprises at leastone amino acid change relative to the wild-type SIRPα sequence, andwherein the amino acid change increases the affinity of the SIRPαpolypeptide binding to CD47, for example by decreasing the off-rate byat least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold,at least 500-fold, or more.

A high affinity SIRPα reagent comprises the portion of SIRPα that issufficient to bind CD47 at a recognizable affinity, e.g., high affinity,which normally lies between the signal sequence and the transmembranedomain, or a fragment thereof that retains the binding activity. Thehigh affinity SIRPα reagent will usually comprise at least the dl domainof SIRPα with modified amino acid residues to increase affinity.

A SIRPα reagent can be used as a “monomer”, in which the binding domainof SIRPα is used, but where the binding domain is provided as a solublemonomeric protein. In other embodiments, a SIRPα variant of the presentinvention is a fusion protein, e.g., fused in frame with a secondpolypeptide, particularly where the second polypeptide provides formultimerization. In some embodiments, the second polypeptide is part orwhole of an immunoglobulin Fc region. The Fc region aids in phagocytosisby providing an “eat me” signal, which enhances the block of the “don'teat me” signal provided by the high affinity SIRPα reagent. In otherembodiments, the second polypeptide is any suitable polypeptide that issubstantially similar to Fc, e.g., providing increased size,multimerization domains, and/or additional binding or interaction withIg molecules.

The amino acid changes that provide for increased affinity are localizedin the dl domain, and thus high affinity SIRPα reagents comprise a dldomain of human SIRPα, with at least one amino acid change relative tothe wild-type sequence within the dl domain. Such a high affinity SIRPαreagent optionally comprises additional amino acid sequences, forexample antibody Fc sequences; portions of the wild-type human SIRPαprotein other than the dl domain, including without limitation residues150 to 374 of the native protein or fragments thereof, usually fragmentscontiguous with the dl domain; and the like. High affinity SIRPαreagents may be monomeric or multimeric, i.e. dimer, trimer, tetramer,etc.

Anti-CD47 antibodies. In some embodiments, a subject anti-CD47 agent isan antibody that specifically binds CD47 (i.e., an anti-CD47 antibody)and reduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). In someembodiments, a suitable anti-CD47 antibody does not activate CD47 uponbinding, for example an antibody that does not induce apoptosis uponbinding. Non-limiting examples of suitable antibodies include clonesB6H12, 5F9, 8B6, and C3 (for example as described in InternationalPatent Publication WO 2011/143624, herein specifically incorporated byreference). Suitable anti-CD47 antibodies include fully human, humanizedor chimeric versions of such antibodies. Humanized antibodies (e.g.,hu5F9-G4) are especially useful for in vivo applications in humans dueto their low antigenicity. Similarly caninized, felinized, etc.antibodies are especially useful for applications in dogs, cats, andother species respectively. Antibodies of interest include humanizedantibodies, or caninized, felinized, equinized, bovinized, porcinized,etc., antibodies, and variants thereof.

Anti-CD47 antibodies. In some embodiments, a subject anti-CD47 agent isan antibody that specifically binds CD47 (i.e., an anti-CD47 antibody)and reduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). In someembodiments, a suitable anti-CD47 antibody does not activate CD47 uponbinding, for example an antibody that does not induce apoptosis uponbinding. Non-limiting examples of suitable antibodies include clonesB6H12, 5F9, 8B6, and C3 (for example as described in InternationalPatent Publication WO 2011/143624, herein specifically incorporated byreference). The 5F9 antibody comprises CDR sequences (SEQ ID NO:1) 5F9heavy chain CDR1: NYNMH; (SEQ ID NO:2) 5F9 heavy chain CDR2:TIYPGNDDTSYNQKFKD; (SEQ ID NO:3) 5F9 heavy chain CDR3: GGYRAMDY; (SEQ IDNO:4) 5F9 light chain CDR1: RSSQSIVYSNGNTYLG; (SEQ ID NO:5) 5F9 lightchain CDR2: KVSNRFS; (SEQ ID NO:6) 5F9 light chain CDR3: FQGSHVPYT.Suitable anti-CD47 antibodies include fully human, humanized or chimericversions of such antibodies. Humanized antibodies (e.g., hu5F9-G4) areespecially useful for in vivo applications in humans due to their lowantigenicity. Similarly caninized, felinized, etc. antibodies areespecially useful for applications in dogs, cats, and other speciesrespectively. Antibodies of interest include humanized antibodies, orcaninized, felinized, equinized, bovinized, porcinized, etc.,antibodies, and variants thereof.

Soluble CD47 polypeptides. In some embodiments, a subject anti-CD47agent is a soluble CD47 polypeptide that specifically binds SIRPα andreduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). A suitablesoluble CD47 polypeptide can bind SIRPα without activating orstimulating signaling through SIRPα because activation of SIRPα wouldinhibit phagocytosis. Instead, suitable soluble CD47 polypeptidesfacilitate the preferential phagocytosis of infected cells overnon-infected cells. Those cells that express higher levels of CD47(e.g., infected cells) relative to normal, non-target cells (normalcells) will be preferentially phagocytosed. Thus, a suitable solubleCD47 polypeptide specifically binds SIRPα without activating/stimulatingenough of a signaling response to inhibit phagocytosis.

In some cases, a suitable soluble CD47 polypeptide can be a fusionprotein (for example as structurally described in US Patent PublicationUS20100239579, herein specifically incorporated by reference). However,only fusion proteins that do not activate/stimulate SIRPα are suitablefor the methods provided herein. Suitable soluble CD47 polypeptides alsoinclude any peptide or peptide fragment comprising variant or naturallyexisting CD47 sequences (e.g., extracellular domain sequences orextracellular domain variants) that can specifically bind SIRPα andinhibit the interaction between CD47 and SIRPα without stimulatingenough SIRPα activity to inhibit phagocytosis.

In certain embodiments, soluble CD47 polypeptide comprises theextracellular domain of CD47, including the signal peptide, such thatthe extracellular portion of CD47 is typically 142 amino acids inlength, and has the amino acid sequence set forth in SEQ ID NO:3. Thesoluble CD47 polypeptides described herein also include CD47extracellular domain variants that comprise an amino acid sequence atleast 65%-75%, 75%-80%, 80-85%, 85%-90%, or 95%-99% (or any percentidentity not specifically enumerated between 65% to 100%), whichvariants retain the capability to bind to SIRPα without stimulatingSIRPα signaling.

In certain embodiments, the signal peptide amino acid sequence may besubstituted with a signal peptide amino acid sequence that is derivedfrom another polypeptide (e.g., for example, an immunoglobulin orCTLA4). For example, unlike full-length CD47, which is a cell surfacepolypeptide that traverses the outer cell membrane, the soluble CD47polypeptides are secreted; accordingly, a polynucleotide encoding asoluble CD47 polypeptide may include a nucleotide sequence encoding asignal peptide that is associated with a polypeptide that is normallysecreted from a cell.

In other embodiments, the soluble CD47 polypeptide comprises anextracellular domain of CD47 that lacks the signal peptide. In anexemplary embodiment, the CD47 extracellular domain lacking the signalpeptide has the amino acid sequence set forth in SEQ ID NO:1 (124 aminoacids). As described herein, signal peptides are not exposed on the cellsurface of a secreted or transmembrane protein because either the signalpeptide is cleaved during translocation of the protein or the signalpeptide remains anchored in the outer cell membrane (such a peptide isalso called a signal anchor). The signal peptide sequence of CD47 isbelieved to be cleaved from the precursor CD47 polypeptide in vivo.

In other embodiments, a soluble CD47 polypeptide comprises a CD47extracellular domain variant. Such a soluble CD47 polypeptide retainsthe capability to bind to SIRPα without stimulating SIRPα signaling. TheCD47 extracellular domain variant may have an amino acid sequence thatis at least 65%-75%, 75%-80%, 80-85%, 85%-90%, or 95%-99% identical(which includes any percent identity between any one of the describedranges) to a reference human CD47 sequence.

The term “antibody” or “antibody moiety” is intended to include anypolypeptide chain-containing molecular structure with a specific shapethat fits to and recognizes an epitope, where one or more non-covalentbinding interactions stabilize the complex between the molecularstructure and the epitope. Antibodies utilized in the present inventionmay be polyclonal antibodies, although monoclonal antibodies arepreferred because they may be reproduced by cell culture orrecombinantly, and can be modified to reduce their antigenicity.

Polyclonal antibodies can be raised by a standard protocol by injectinga production animal with an antigenic composition. See, e.g., Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,1988. When utilizing an entire protein, or a larger section of theprotein, antibodies may be raised by immunizing the production animalwith the protein and a suitable adjuvant (e.g., Freund's, Freund'scomplete, oil-in-water emulsions, etc.) When a smaller peptide isutilized, it is advantageous to conjugate the peptide with a largermolecule to make an immunostimulatory conjugate. Commonly utilizedconjugate proteins that are commercially available for such use includebovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). In orderto raise antibodies to particular epitopes, peptides derived from thefull sequence may be utilized. Alternatively, in order to generateantibodies to relatively short peptide portions of the protein target, asuperior immune response may be elicited if the polypeptide is joined toa carrier protein, such as ovalbumin, BSA or KLH. Alternatively, formonoclonal antibodies, hybridomas may be formed by isolating thestimulated immune cells, such as those from the spleen of the inoculatedanimal. These cells are then fused to immortalized cells, such asmyeloma cells or transformed cells, which are capable of replicatingindefinitely in cell culture, thereby producing an immortal,immunoglobulin-secreting cell line. In addition, the antibodies orantigen binding fragments may be produced by genetic engineering.Humanized, chimeric, or xenogeneic human antibodies, which produce lessof an immune response when administered to humans, are preferred for usein the present invention.

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)2, or other fragments) are useful as antibodymoieties in the present invention. Such antibody fragments may begenerated from whole immunoglobulins by ricin, pepsin, papain, or otherprotease cleavage. “Fragment,” or minimal immunoglobulins may bedesigned utilizing recombinant immunoglobulin techniques. For instance“Fv” immunoglobulins for use in the present invention may be produced bylinking a variable light chain region to a variable heavy chain regionvia a peptide linker (e.g., poly-glycine or another sequence which doesnot form an alpha helix or beta sheet motif).

Antibodies include free antibodies and antigen binding fragments derivedtherefrom, and conjugates, e.g. pegylated antibodies, drug,radioisotope, or toxin conjugates, and the like. Monoclonal antibodiesdirected against a specific epitope, or combination of epitopes, willallow for the targeting and/or depletion of cellular populationsexpressing the marker. Various techniques can be utilized usingmonoclonal antibodies to screen for cellular populations expressing themarker(s), and include magnetic separation using antibody-coatedmagnetic beads, “panning” with antibody attached to a solid matrix(i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660;and Morrison et al. Cell, 96:737-49 (1999)). These techniques allow forthe screening of particular populations of cells; inimmunohistochemistry of biopsy samples; in detecting the presence ofmarkers shed by cancer cells into the blood and other biologic fluids,and the like.

Humanized versions of such antibodies are also within the scope of thisinvention. Humanized antibodies are especially useful for in vivoapplications in humans due to their low antigenicity.

The phrase “multispecific or bispecific antibody” refers to a syntheticor recombinant antibody that recognizes more than one protein.Bispecific antibodies directed against a combination of epitopes, willallow for the targeting and/or depletion of cellular populationsexpressing the combination of epitopes. Exemplary bi-specific antibodiesinclude those targeting a combination of CD47 and an SCLC cancer cellmarker. Generation of bi-specific antibodies are described in theliterature, for example, in U.S. Pat. Nos. 5,989,830, 5,798,229, whichare incorporated herein by reference. Higher order specificities, e.g.trispecific antibodies, are described by Holliger and Hudson (2005)Nature Biotechnology 23:1126-1136.

The efficacy of a CD47 inhibitor can be assessed by assaying CD47activity. The above-mentioned assays or modified versions thereof areused. In an exemplary assay, SCLC are incubated with bone marrow derivedmacrophages, in the presence or absence of the candidate agent. Aninhibitor of the cell surface CD47 will up-regulate phagocytosis by atleast about 10%, or up to 20%, or 50%, or 70% or 80% or up to about 90%compared to the phagocytosis in absence of the candidate agent.Similarly, an in vitro assay for levels of tyrosine phosphorylation ofSIRPα will show a decrease in phosphorylation by at least about 10%, orup to 20%, or 50%, or 70% or 80% or up to about 90% compared tophosphorylation observed in absence of the candidate agent.

In one embodiment of the invention, the agent, or a pharmaceuticalcomposition comprising the agent, is provided in an amount effective todetectably inhibit the binding of CD47 to SIRPα receptor present on thesurface of phagocytic cells. The effective amount is determined viaempirical testing routine in the art. The effective amount may varydepending on the number of cells being transplanted, site oftransplantation and factors specific to the transplant recipient.

The terms “phagocytic cells” and “phagocytes” are used interchangeablyherein to refer to a cell that is capable of phagocytosis. There arefour main categories of phagocytes: macrophages, mononuclear cells(histiocytes and monocytes); polymorphonuclear leukocytes (neutrophils)and dendritic cells.

The term “biological sample” encompasses a variety of sample typesobtained from an organism and can be used in a diagnostic or monitoringassay. The term encompasses blood and other liquid samples of biologicalorigin, solid tissue samples, such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. The termencompasses samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components. The term encompasses a clinicalsample, and also includes cells in cell culture, cell supernatants, celllysates, serum, plasma, biological fluids, and tissue samples.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, e.g. mouse, rat, rabbit, pig, primate, including humans andother apes, particularly a human, and includes: (a) preventing thedisease or symptom from occurring in a subject which may be predisposedto the disease or symptom but has not yet been diagnosed as having it;(b) inhibiting the disease symptom, i.e., arresting its development; or(c) relieving the disease symptom, i.e., causing regression of thedisease or symptom.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,used interchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

A “host cell”, as used herein, refers to a microorganism or a eukaryoticcell or cell line cultured as a unicellular entity which can be, or hasbeen, used as a recipient for a recombinant vector or other transferpolynucleotides, and include the progeny of the original cell which hasbeen transfected. It is understood that the progeny of a single cell maynot necessarily be completely identical in morphology or in genomic ortotal DNA complement as the original parent, due to natural, accidental,or deliberate mutation.

The terms “cancer”, “neoplasm”, “tumor”, and “carcinoma”, are usedinterchangeably herein to refer to cells which exhibit relativelyautonomous growth, so that they exhibit an aberrant growth phenotypecharacterized by a significant loss of control of cell proliferation. Ingeneral, cells of interest for detection or treatment in the presentapplication include precancerous (e.g., benign), malignant,pre-metastatic, metastatic, and non-metastatic cells. Detection ofcancerous cells is of particular interest. The term “normal” as used inthe context of “normal cell,” is meant to refer to a cell of anuntransformed phenotype or exhibiting a morphology of a non-transformedcell of the tissue type being examined. “Cancerous phenotype” generallyrefers to any of a variety of biological phenomena that arecharacteristic of a cancerous cell, which phenomena can vary with thetype of cancer. The cancerous phenotype is generally identified byabnormalities in, for example, cell growth or proliferation (e.g.,uncontrolled growth or proliferation), regulation of the cell cycle,cell mobility, cell-cell interaction, or metastasis, etc.

“Therapeutic target” refers to a gene or gene product that, uponmodulation of its activity (e.g., by modulation of expression,biological activity, and the like), can provide for modulation of thecancerous phenotype. As used throughout, “modulation” is meant to referto an increase or a decrease in the indicated phenomenon (e.g.,modulation of a biological activity refers to an increase in abiological activity or a decrease in a biological activity).

Lung Cancer

Lung carcinoma is the leading cause of cancer-related death worldwide.About 85% of cases are related to cigarette smoking. Symptoms caninclude cough, chest discomfort or pain, weight loss, and, lesscommonly, hemoptysis; however, many patients present with metastaticdisease without any clinical symptoms. The diagnosis is typically madeby chest x-ray or CT and confirmed by biopsy. Depending on the stage ofthe disease, treatment includes surgery, chemotherapy, radiationtherapy, or a combination. For the past several decades, the prognosisfor a lung cancer patient has been poor, particularly for patients withstage IV (metastatic) disease.

Respiratory epithelial cells require prolonged exposure tocancer-promoting agents and accumulation of multiple genetic mutationsbefore becoming neoplastic (an effect called field carcinogenesis). Insome patients with lung cancer, secondary or additional mutations ingenes that stimulate cell growth (K-ras, MYC), cause abnormalities ingrowth factor receptor signaling (EGFR, HER2/neu), and inhibit apoptosiscontribute to proliferation of abnormal cells. In addition, mutationsthat inhibit tumor-suppressor genes (p53, APC) can lead to cancer. Othermutations that may be responsible include the EML-4-ALK translocationand mutations in ROS-1, BRAF, and PI3KCA. Genes such as these that areprimarily responsible for lung cancer are called driver mutations.Although driver mutations can cause or contribute to lung cancer amongsmokers, these mutations are particularly likely to be a cause of lungcancer among nonsmokers.

Chest x-ray is often the initial imaging test. It may show clearlydefined abnormalities, such as a single mass or multifocal masses or asolitary pulmonary nodule, an enlarged hilum, widened mediastinum,tracheobronchial narrowing, atelectasis, non-resolving parenchymalinfiltrates, cavitary lesions, or unexplained pleural thickening oreffusion. These findings are suggestive but not diagnostic of lungcancer and require follow-up with CT scans or combined PET-CT scans andcytopathologic confirmation.

CT shows many characteristic anatomic patterns and appearances that maystrongly suggest the diagnosis. CT also can guide core needle biopsy ofaccessible lesions and is useful for staging. If a lesion found on aplain x-ray is highly likely to be lung cancer, PET-CT may be done. Thisstudy combines anatomic imaging from CT with functional imaging fromPET. The PET images can help differentiate inflammatory and malignantprocesses.

SCLC has 2 stages: limited and extensive. Limited-stage SCLC disease iscancer confined to one hemithorax (including ipsilateral lymph nodes)that can be encompassed within one tolerable radiation therapy port,unless there is a pleural or pericardial effusion. Extensive-stagedisease is cancer outside a single hemithorax or the presence ofmalignant cells detected in pleural or pericardial effusions. Less thanone third of patients with SCLC will present with limited-stage disease;the remainder of patients often have extensive distant metastases. Theoverall prognosis for SCLC is poor. The median survival time forlimited-stage SCLC is 20 mo, with a 5-yr survival rate of 20%. Patientswith extensive-stage SCLC do especially poorly, with a 5-yr survivalrate of <1%.

NSCLC has 4 stages, I through IV (using the TNM system). TNM staging isbased on tumor size, tumor and lymph node location, and the presence orabsence of distant metastases. The 5-yr survival rate of patients withNSCLC varies by stage, from 60 to 70% for patients with stage I diseaseto <1% for patients with stage IV disease.

Conventional treatment varies by cell type and by stage of disease. Manypatient factors not related to the tumor affect treatment choice. Poorcardiopulmonary reserve, undernutrition, frailty or poor physicalperformance status, comorbidities, including cytopenias, and psychiatricor cognitive illness all may lead to a decision for palliative overcurative treatment or for no treatment at all, even though a cure withaggressive therapy might technically be possible.

SCLC of any stage is typically initially responsive to treatment, butresponses are usually short-lived. Chemotherapy, with or withoutradiation therapy, is given depending on the stage of disease. In manypatients, chemotherapy prolongs survival and improves quality of lifeenough to warrant its use. Surgery generally plays no role in treatmentof SCLC, although it may be curative in the rare patient who has a smallfocal tumor without spread (such as a solitary pulmonary nodule) whounderwent surgical resection before the tumor was identified as SCLC.Chemotherapy regimens of etoposide and a platinum compound (eithercisplatin or carboplatin) are commonly used, as are other drugs, such asirinotecan, topotecan, vinca alkaloids (vinblastine, vincristine,vinorelbine), alkylating agents (cyclophosphamide, ifosfamide),doxorubicin, taxanes (docetaxel, paclitaxel), and gemcitabine. Whendisease is confined to a hemithorax, radiation therapy further improvesclinical outcomes; such response to radiation therapy was the basis forthe definition of limited-stage disease. The use of cranial radiation toprevent brain metastases is also advocated in certain cases;micrometastases are common in SCLC, and chemotherapy has less ability tocross the blood-brain barrier.

In extensive-stage disease, treatment is based on chemotherapy ratherthan radiation therapy, although radiation therapy is often used aspalliative treatment for metastases to bone or brain. In patients withan excellent response to chemotherapy, prophylactic brain irradiation issometimes used as in limited-stage SCLC to prevent growth of SCLC in thebrain.

Treatment for NSCLC typically involves assessment of eligibility forsurgery followed by choice of surgery, chemotherapy, radiation therapy,or a combination of modalities as appropriate, depending on tumor typeand stage.

Treatment of Cancer

The invention provides methods for reducing growth of lung cancer cellsthrough the introduction of an effective dose of a targeted therapeuticagent directed to a lung cancer cell surface marker, including withoutlimitation CD24, CD166, CD56, CD326, CD298, CD29, CD63, CD9, CD164,CD99, CD46, CD59, CD57, CD165, EpCAM, etc. In some embodiments themarker is one of CD56, CD44, CD99 and EpCam. In preferred embodimentsthe targeted therapeutic agent is combined with a CD47 blocking agent,e.g. soluble SIRPα monomer or multimer, an anti-CD47 antibody, smallmolecule, etc. In certain embodiments the cancer is SCLC. By blockingthe activity of CD47, the downregulation of phagocytosis that is foundwith certain tumor cells is prevented.

“Reducing growth of cancer cells” includes, but is not limited to,reducing proliferation of cancer cells, and reducing the incidence of anon-cancerous cell becoming a cancerous cell. Whether a reduction incancer cell growth has been achieved can be readily determined using anyknown assay, including, but not limited to, [³H]-thymidineincorporation; counting cell number over a period of time; detectingand/or measuring a marker associated with SCLC, etc.

Whether a substance, or a specific amount of the substance, is effectivein treating cancer can be assessed using any of a variety of knowndiagnostic assays for cancer, including, but not limited to biopsy,contrast radiographic studies, CAT scan, and detection of a tumor markerassociated with cancer in the blood of the individual. The substance canbe administered systemically or locally, usually systemically.

As an alternative embodiment, an agent, e.g. a chemotherapeutic drugthat reduces cancer cell growth, can be targeted to a cancer cell byconjugation to a CD47 specific antibody. Thus, in some embodiments, theinvention provides a method of delivering a drug to a cancer cell,comprising administering a drug-antibody complex to a subject, whereinthe antibody is specific for a cancer-associated polypeptide, and thedrug is one that reduces cancer cell growth, a variety of which areknown in the art. Targeting can be accomplished by coupling (e.g.,linking, directly or via a linker molecule, either covalently ornon-covalently, so as to form a drug-antibody complex) a drug to anantibody specific for a cancer-associated polypeptide. Methods ofcoupling a drug to an antibody are well known in the art and need not beelaborated upon herein.

In certain embodiments, a bi-specific antibody may be used. For examplea bi-specific antibody in which one antigen binding domain is directedagainst CD47 and the other antigen binding domain is directed against acancer cell marker, such as CD24, CD166, CD56, CD326, CD298, CD29, CD63,CD9, CD164, CD99, CD46, CD59, CD57, CD165, EpCAM, etc. may be used.

Generally, as the term is utilized in the specification, “antibody” or“antibody moiety” is intended to include any polypeptidechain-containing molecular structure that has a specific shape whichfits to and recognizes an epitope, where one or more non-covalentbinding interactions stabilize the complex between the molecularstructure and the epitope. For monoclonal antibodies, hybridomas may beformed by isolating the stimulated immune cells, such as those from thespleen of the inoculated animal. These cells are then fused toimmortalized cells, such as myeloma cells or transformed cells, whichare capable of replicating indefinitely in cell culture, therebyproducing an immortal, immunoglobulin-secreting cell line. The immortalcell line utilized is preferably selected to be deficient in enzymesnecessary for the utilization of certain nutrients. Many such cell lines(such as myelomas) are known to those skilled in the art, and include,for example: thymidine kinase (TK) or hypoxanthine-guaninephosphoriboxyl transferase (HGPRT). These deficiencies allow selectionfor fused cells according to their ability to grow on, for example,hypoxanthine aminopterinthymidine medium (HAT).

Antibodies which have a reduced propensity to induce a violent ordetrimental immune response in humans (such as anaphylactic shock), andwhich also exhibit a reduced propensity for priming an immune responsewhich would prevent repeated dosage with the antibody therapeutic orimaging agent (e.g., the human-anti-murine-antibody “HAMA” response),are preferred for use in the invention. These antibodies are preferredfor all administrative routes. Thus, humanized, chimeric, or xenogenichuman antibodies, which produce less of an immune response whenadministered to humans, are preferred for use in the present invention.

Chimeric antibodies may be made by recombinant means by combining themurine variable light and heavy chain regions (VK and VH), obtained froma murine (or other animal-derived) hybridoma clone, with the humanconstant light and heavy chain regions, in order to produce an antibodywith predominantly human domains. The production of such chimericantibodies is well known in the art, and may be achieved by standardmeans (as described, e.g., in U.S. Pat. No. 5,624,659, incorporatedfully herein by reference). Humanized antibodies are engineered tocontain even more human-like immunoglobulin domains, and incorporateonly the complementarity-determining regions of the animal-derivedantibody. This is accomplished by carefully examining the sequence ofthe hyper-variable loops of the variable regions of the monoclonalantibody, and fitting them to the structure of the human antibodychains. Although facially complex, the process is straightforward inpractice. See, e.g., U.S. Pat. No. 6,187,287, incorporated fully hereinby reference.

Alternatively, polyclonal or monoclonal antibodies may be produced fromanimals which have been genetically altered to produce humanimmunoglobulins. The transgenic animal may be produced by initiallyproducing a “knock-out” animal which does not produce the animal'snatural antibodies, and stably transforming the animal with a humanantibody locus (e.g., by the use of a human artificial chromosome). Onlyhuman antibodies are then made by the animal. Techniques for generatingsuch animals, and deriving antibodies therefrom, are described in U.S.Pat. Nos. 6,162,963 and 6,150,584, incorporated fully herein byreference. Such fully human xenogenic antibodies are a preferredantibody for use in the methods and compositions of the presentinvention. Alternatively, single chain antibodies can be produced fromphage libraries containing human variable regions. See U.S. Pat. No.6,174,708, incorporated fully herein by reference.

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) are useful as antibodymoieties in the present invention. Such antibody fragments may begenerated from whole immunoglobulins by ficin, pepsin, papain, or otherprotease cleavage. “Fragment,” or minimal immunoglobulins may bedesigned utilizing recombinant immunoglobulin techniques. For instance“Fv” immunoglobulins for use in the present invention may be produced bylinking a variable light chain region to a variable heavy chain regionvia a peptide linker (e.g., poly-glycine or another sequence which doesnot form an alpha helix or beta sheet motif).

Fv fragments are heterodimers of the variable heavy chain domain (VH)and the variable light chain domain (VL). The heterodimers of heavy andlight chain domains that occur in whole IgG, for example, are connectedby a disulfide bond. Recombinant Fvs in which VH and VL are connected bya peptide linker are typically stable, see, for example, Huston et al.,Proc. Natl. Acad, Sci. USA 85:5879 5883 (1988) and Bird et al., Science242:423 426 (1988), both fully incorporated herein, by reference. Theseare single chain Fvs which have been found to retain specificity andaffinity and have been shown to be useful for imaging tumors and to makerecombinant immunotoxins for tumor therapy. However, researchers havefound that some of the single chain Fvs have a reduced affinity forantigen and the peptide linker can interfere with binding. Improved Fv'shave been also been made which comprise stabilizing disulfide bondsbetween the VH and VL regions, as described in U.S. Pat. No. 6,147,203,incorporated fully herein by reference. Any of these minimal antibodiesmay be utilized in the present invention, and those which are humanizedto avoid HAMA reactions are preferred for use in embodiments of theinvention.

Derivatized polypeptides with added chemical linkers, detectablemoieties such as fluorescent dyes, enzymes, substrates, chemiluminescentmoieties, specific binding moieties such as streptavidin, avidin, orbiotin, or drug conjugates may be utilized in the methods andcompositions of the present invention.

In some embodiments of the invention, the polypeptide reagents of theinvention are coupled or conjugated to one or more therapeutic,cytotoxic, or imaging moieties. As used herein, “cytotoxic moiety” (C)simply means a moiety which inhibits cell growth or promotes cell deathwhen proximate to or absorbed by the cell. Suitable cytotoxic moietiesin this regard include radioactive isotopes (radionuclides), chemotoxicagents such as differentiation inducers and small chemotoxic drugs,toxin proteins, and derivatives thereof. Agents may be conjugated to apolypeptide reagent of the invention by any suitable technique, withappropriate consideration of the need for pharmokinetic stability andreduced overall toxicity to the patient. A therapeutic agent may becoupled to a suitable moiety either directly or indirectly (e.g. via alinker group). A direct reaction is possible when each possesses afunctional group capable of reacting with the other. For example, anucleophilic group, such as an amino or sulfhydryl group, may be capableof reacting with a carbonyl-containing group, such as an anhydride or anacid halide, or with an alkyl group containing a good leaving group(e.g., a halide). Alternatively, a suitable chemical linker group may beused. A linker group can function as a spacer to distance a polypeptidereagent of the invention from an agent in order to avoid interferencewith binding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on a moiety or a polypeptidereagent of the invention, and thus increase the coupling efficiency. Anincrease in chemical reactivity may also facilitate the use of moieties,or functional groups on moieties, which otherwise would not be possible.

Suitable linkage chemistries include maleimidyl linkers and alkyl halidelinkers (which react with a sulfhydryl on the antibody moiety) andsuccinimidyl linkers (which react with a primary amine on the antibodymoiety). Several primary amine and sulfhydryl groups are present onimmunoglobulins, and additional groups may be designed into recombinantimmunoglobulin molecules. It will be evident to those skilled in the artthat a variety of bifunctional or polyfunctional reagents, both homo-and hetero-functional (such as those described in the catalog of thePierce Chemical Co., Rockford, Ill.), may be employed as a linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958. As an alternative coupling method, cytotoxic moieties may becoupled to a polypeptide reagent of the invention through a an oxidizedcarbohydrate group at a glycosylation site, as described in U.S. Pat.Nos. 5,057,313 and 5,156,840. Yet another alternative method of couplinga polypeptide reagent of the invention to a cytotoxic or therapeuticmoiety is by the use of a non-covalent binding pair, such asstreptavidin/biotin, or avidin/biotin. In these embodiments, one memberof the pair is covalently coupled to the anti-CD47, CV1, etc. moiety andthe other member of the binding pair is covalently coupled to thetherapeutic, cytotoxic, or imaging moiety.

Where a cytotoxic moiety is more potent when free from the bindingportion of a polypeptide reagent of the invention, it may be desirableto use a linker group which is cleavable during or upon internalizationinto a cell, or which is gradually cleavable over time in theextracellular environment. A number of different cleavable linker groupshave been described. The mechanisms for the intracellular release of acytotoxic moiety agent from these linker groups include cleavage byreduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014), byhydrolysis of derivatized amino acid side chains (e.g., U.S. Pat. No.4,638,045), by serum complement-mediated hydrolysis (e.g., U.S. Pat. No.4,671,958), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789).

It may be desirable to couple more than one moiety to a polypeptidereagent of the invention. By poly-derivatizing the reagent, severalstrategies may be simultaneously implemented, e.g. a therapeuticantibody may be labeled for tracking by a visualization technique.Regardless of the particular embodiment, conjugates with more than onemoiety may be prepared in a variety of ways. For example, more than onemoiety may be coupled directly to a polypeptide molecule, or linkerswhich provide multiple sites for attachment (e.g., dendrimers) can beused. Alternatively, a carrier with the capacity to hold more than onecytotoxic or imaging moiety can be used.

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group, and non-covalentassociations. Suitable covalent-bond carriers include proteins such asalbumins (e.g., U.S. Pat. No. 4,507,234), peptides, and polysaccharidessuch as aminodextran (e.g., U.S. Pat. No. 4,699,784), each of which havemultiple sites for the attachment of moieties. A carrier may also bearan agent by non-covalent associations, such as non-covalent bonding orby encapsulation, such as within a liposome vesicle (e.g., U.S. Pat.Nos. 4,429,008 and 4,873,088). Encapsulation carriers are especiallyuseful for imaging moiety conjugation to antibody moieties for use inthe invention, as a sufficient amount of the imaging moiety (dye,magnetic resonance contrast reagent, etc.) for detection may be moreeasily associated with the antibody moiety. In addition, encapsulationcarriers are also useful in chemotoxic therapeutic embodiments, as theycan allow the therapeutic compositions to gradually release a chemotoxicmoiety over time while concentrating it in the vicinity of the tumorcells.

Preferred radionuclides for use as cytotoxic moieties are radionuclideswhich are suitable for pharmacological administration. Suchradionuclides include ¹²³I, ¹²⁵I, ¹³¹I, ⁹⁰Y, ²¹¹At, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re,²¹²Pb, and ²¹²Bi. Iodine and astatine isotopes are more preferredradionuclides for use in the therapeutic compositions of the presentinvention, as a large body of literature has been accumulated regardingtheir use.

Preferred chemotoxic agents include small-molecule drugs such ascarboplatin, cisplatin, vincristine, taxanes such as paclitaxel anddoceltaxel, hydroxyurea, gemcitabine, vinorelbine, irinotecan,tirapazamine, matrilysin, methotrexate, pyrimidine and purine analogs,and other suitable small toxins known in the art. Preferred chemotoxindifferentiation inducers include phorbol esters and butyric acid.Chemotoxic moieties may be directly conjugated to the antibody moietyvia a chemical linker, or may be encapsulated in a carrier, which is inturn coupled to the antibody. Preferred toxin proteins for use ascytotoxic moieties include ricins A and B, abrin, diphtheria toxin,bryodin 1 and 2, momordin, trichokirin, cholera toxin, gelonin,Pseudomonas exotoxin, Shigella toxin, pokeweed antiviral protein, andother toxin proteins known in the medicinal biochemistry arts. As thesetoxin agents may elicit undesirable immune responses in the patient,especially if injected intravascularly, it is preferred that they beencapsulated in a carrier for coupling to the antibody.

For administration, a targeted therapeutic agent, or combination oftargeted therapeutic agents may be administered separately or together;and will generally be administered within the same general time frame,e.g. within a week, within 3-4 days, within 1 day or simultaneously witheach other.

The agent or agents are mixed, prior to administration, with anon-toxic, pharmaceutically acceptable carrier substance. Usually, thiswill be an aqueous solution, such as normal saline or phosphate-bufferedsaline (PBS), Ringer's solution, lactate-Ringer's solution, or anyisotonic physiologically acceptable solution for administration by thechosen means. Preferably, the solution is sterile and pyrogen-free, andis manufactured and packaged under current Good Manufacturing Processes(GMPs), as approved by the FDA. The clinician of ordinary skill isfamiliar with appropriate ranges for pH, tonicity, and additives orpreservatives when formulating pharmaceutical compositions foradministration by intravascular injection, direct injection into thelymph nodes, intraperitoneal, or by other routes. In addition toadditives for adjusting pH or tonicity, the agents may be stabilizedagainst aggregation and polymerization with amino acids and non-ionicdetergents, polysorbate, and polyethylene glycol. Optionally, additionalstabilizers may include various physiologically-acceptable carbohydratesand salts. Also, polyvinylpyrrolidone may be added in addition to theamino acid. Suitable therapeutic immunoglobulin solutions which arestabilized for storage and administration to humans are described inU.S. Pat. No. 5,945,098, incorporated fully herein by reference. Otheragents, such as human serum albumin (HSA), may be added to thetherapeutic or imaging composition to stabilize the antibody conjugates.

The compositions of the invention may be administered using anymedically appropriate procedure, e.g., intravascular (intravenous,intraarterial, intracapillary) administration, injection into the tumor,etc. Intravascular injection may be by intravenous or intraarterialinjection. The effective amount of the therapeutic compositions to begiven to a particular patient will depend on a variety of factors,several of which will be different from patient to patient. A competentclinician will be able to determine an effective amount of a therapeuticcomposition to administer to a patient to retard the growth and promotethe death of tumor cells. Dosage of the agents will depend on thetreatment of the tumor, route of administration, the nature of thetherapeutics, sensitivity of the tumor to the therapeutics, etc.Utilizing LD₅₀ animal data, and other information available for theconjugated cytotoxic or imaging moiety, a clinician can determine themaximum safe dose for an individual, depending on the route ofadministration. For instance, an intravenously administered dose may bemore than an locally administered dose, given the greater body of fluidinto which the therapeutic composition is being administered. Similarly,compositions which are rapidly cleared from the body may be administeredat higher doses, or in repeated doses, in order to maintain atherapeutic concentration. Utilizing ordinary skill, the competentclinician will be able to optimize the dosage of a particulartherapeutic or imaging composition in the course of routine clinicaltrials.

Typically an effective dosage will be 0.001 to 100 milligrams ofantibody per kilogram subject body weight. The ratio of anti-CD47 to thesecond agent may range from 1:100; 1:50; 1:10; 1:5; 1:2; 1:1; 2:1; 5:1;10:1; 50:1; 100:1. The agents can be administered to the subject in aseries of more than one administration. For therapeutic compositions,regular periodic administration (e.g., every 2-3 days) will sometimes berequired, or may be desirable to reduce toxicity. For therapeuticcompositions which will be utilized in repeated-dose regimens, antibodymoieties which do not provoke HAMA or other immune responses arepreferred.

Example 1 High-Affinity SIRPα Variants Enhance Macrophage Destruction ofSmall Cell Lung Cancer

CD47 allows cancer cells to evade the immune system by signaling throughSIRPα, an inhibitory receptor on macrophages. We recently developednext-generation CD47 antagonists by engineering the N-terminalimmunoglobulin domain of SIRPα. These “high-affinity SIRPα variants”have an affinity for human CD47 (K_(D)) as low as 11.1 pM, approximately50,000-fold improved over wild-type SIRPα. When combined withtumor-specific antibodies, the high-affinity SIRPα variants act asimmunotherapeutic adjuvants to maximize macrophage destruction of cancercells.

We have now applied these reagents to small cell lung cancer (SCLC), acancer with poor prognosis for which no clinically approved antibodiesexist. We found SCLC cell lines and primary samples expressed highlevels of CD47 on their surface. Using human macrophages, we found thatCD47-blocking therapies were able to induce macrophage phagocytosis ofSCLC cells. Treatment of mice bearing primary human SCLC tumors withCD47-blocking antibodies was able to inhibit tumor growth andsignificantly prolong survival. To identify novel SCLC antigens that canbe targeted in combination with high-affinity SIRPα variants, SCLCsamples were screened by flow cytometry using comprehensive antibodyarrays.

We validated tumor-specific antigens on the surface of SCLC cells, andidentified antibodies to these antigens that could stimulatephagocytosis in vitro. When combined with high-affinity SIRPα monomers,the ability of these antibodies to stimulate phagocytosis wasdramatically enhanced.

Example 2 CD47-Blocking Therapies Stimulate Macrophage Destruction ofSmall Cell Lung Cancer

Small cell lung cancer (SCLC) is a highly aggressive subtype of lungcancer with dismal prognosis. There are no clinically approvedantibodies, targeted therapies, or immunotherapies for the disease. Wefound that SCLC samples expressed high levels of CD47, a cell-surfacemolecule that allows cancer cells to evade the immune system. Inparticular, CD47 promotes immune evasion by signaling through SIRPα, aninhibitory receptor on macrophages. We hypothesized that CD47-blockingtherapies could be applied to the treatment of SCLC. We found thatCD47-blocking therapies were able to induce macrophage phagocytosis ofSCLC samples in vitro. CD47-blocking therapies also inhibited tumorgrowth and significantly prolonged survival of mice bearing SCLC tumors.Furthermore, using comprehensive antibody arrays, we identified severalnew and established therapeutic targets on the surface of SCLC cells.Antibodies to these targets could elicit macrophage phagocytosis andwere enhanced when combined with CD47-blocking therapies. These findingssuggest that therapies that disrupt the CD47-SIRPα axis could benefitpatients with SCLC, particularly when combined with tumor-specificantibodies.

Small cell lung cancer (SCLC), which derives from neuroendocrine cellsof the lung, is one of the most lethal subtypes of cancer in humans.Each year, more than 25,000 patients are diagnosed with SCLC in theUnited States alone, and patients typically live only 6-12 months afterdiagnosis. The 5-year survival rate has remained dismal, hovering around5% since the 1970s. Except for the combination of radiation andchemotherapy, there have been no new therapeutic approaches implementedin the past 30 years. Despite a plethora of clinical trials, no targetedtherapies have been approved for SCLC. SCLC is strongly linked to heavycigarette smoking, and increased smoking rates in developing countrieswill continue to increase the worldwide prevalence of SCLC in thefuture. For these reasons, there is a need to identify novel therapeutictargets and generate new treatments for patients with SCLC.

One of the most promising advances in the field of oncology isimmunotherapy, which aims to stimulate a patient's own immune system toattack and eliminate cancer. As tumors develop, they acquire mechanismsto avoid destruction by the immune system. By understanding thesemechanisms, we can develop new strategies to coax the immune system torecognize cancer as foreign. Previous studies have identified CD47, acell-surface molecule, as a “marker of self” that prevents cells of theinnate immune system from attacking hematologic malignancies and certaintypes of solid tumors. CD47 acts by sending inhibitory signals throughSIRPα, a receptor expressed on the surface of macrophages and othermyeloid cells. In this sense, the CD47-SIRPα interaction represents amyeloid-specific immune checkpoint. A number of reagents have beengenerated to disrupt signaling by the CD47-SIRPα axis, includinganti-CD47 antibodies and engineered variants of its receptor, SIRPα.Recent studies have shown that blockade of CD47 lowers the threshold formacrophage phagocytosis of cancer. We hypothesized that SCLC cells alsoexpress CD47 and that CD47-blocking therapies could be used to stimulatemacrophage phagocytosis of SCLC cells and inhibit growth of SCLC tumorsin vivo.

Furthermore, CD47-blocking therapies have been shown to enhance theresponse of macrophages to monoclonal antibodies. Monoclonalantibodies—such as rituximab for lymphoma or trastuzumab for Her2⁺breast cancer—have demonstrated immense success for the treatment ofcancer. No monoclonal antibodies are clinically approved for thetreatment of SCLC, thus, we aimed to identify new SCLC surface antigensthat could be targeted with monoclonal antibodies. While treatment withmonoclonal antibodies can produce robust anti-tumor effects, they oftenfail to elicit cures when used as single agents, highlighting the needto improve the efficacy of these approaches. Therefore, we aimed tocombine CD47-blocking therapies with other antibodies to achieve maximalanti-tumor responses against SCLC.

As a first step in our approach, we investigated whether CD47 wasexpressed on the surface of SCLC samples. Next, we examined whetherCD47-blocking therapies could stimulate macrophage phagocytosis of SCLCin vitro. Mouse models of human cancer were used to evaluate theresponse of SCLC samples to CD47-blocking therapies in vivo. To identifynew therapeutic targets on the surface of SCLC samples, we performedhigh-throughput flow cytometry using comprehensive antibody arrays.Last, we aimed to demonstrate that antibodies towards the identifiedantigens could be combined with CD47-blocking therapies to furtherincrease phagocytosis. The overall objectives of this study were tovalidate CD47-blocking therapies for SCLC and identify additionalantibodies that could be used to target SCLC. In this manner, we aim toidentify new immunotherapeutic combinations that could be used for thebenefit of patients with SCLC.

Results

CD47 is Expressed on the Surface of SCLC. To evaluate whetherCD47-blocking therapies could be applied to SCLC, we first examinedexpression of CD47 on the surface of SCLC cells. We obtained six SCLCcell lines and subjected them to flow cytometry to evaluate CD47expression on the cell surface. All six cell lines exhibited high CD47expression (FIG. 5A). We also evaluated CD47 surface expression on aSCLC patient-derived xenograft obtained from a primary SCLC patientsample. Similar to the cell lines, the H29 patient sample also expressedhigh levels of CD47 on its surface (FIG. 5B). These findings suggestedthat CD47 is an immunotherapeutic target on SCLC.

CD47-blocking Antibodies Induce Phagocytosis of SCLC by HumanMacrophages. To validate CD47 as a genuine therapeutic target on SCLC,we performed in vitro phagocytosis assays using human macrophages andSCLC samples. Macrophages were co-cultured with SCLC cells in thepresence of a vehicle control or anti-CD47 antibodies. We testedanti-CD47 antibody clone Hu5F9-G4, a humanized anti-CD47 antibody thatblocks the interaction between CD47 and SIRPα and is under investigationin a Phase I clinical trial for solid tumors (ClinicalTrials.govidentifier: NCT02216409). High-throughput flow cytometry was used tomeasure phagocytosis, which was evaluated by the percentage ofmacrophages engulfing calcein AM-labeled SCLC cells (FIGS. 5C and D).Fluorescence-activated cell sorting was used to confirm the doublepositive population contained macrophages with engulfed tumor cells(FIG. 5E). Four SCLC samples were subjected to evaluation inphagocytosis assays. Three cell lines (NCI-H524, NCI-1688, and NCI-H82)exhibited significant increases in phagocytosis when treated with theCD47-blocking antibody (FIG. 5F). One cell line, NCI-H196, appeared tobe resistant to phagocytosis, suggesting additional mechanisms modifythe susceptibility of this cell line to macrophage attack. Thepatient-derived xenograft H29 was also subjected to phagocytosis assayswith human macrophages. Treatment of this sample with anti-CD47antibodies also resulted in a significant increase in phagocytosis (FIG.5G).

CD47-blocking Antibodies Inhibit Growth of SCLC Tumors in vivo. Toevaluate the potential of CD47-blocking agents when administered astherapies for human SCLC, we established xenograft models of human SCLC.We engrafted NCI-H82 cells into the lower left flanks of NSG mice, whichlack functional T cells, B cells, and NK cells but retain functionalmacrophages. Approximately one week after engraftment, mice wererandomized into treatment with vehicle control or 250 μg anti-CD47antibody clone Hu5F9-G4 administered every other day. Tumor volumemeasurements were used to evaluate mice for a response to therapy. Aftertwo weeks of treatment, a significant difference in median tumor volumewas observed that persisted through the remainder of the experiment(FIG. 6A). After approximately one month of treatment, the median tumorvolume for the vehicle control cohort was 837.8 mm³ versus 160.2 mm³ forthe cohort treated with the anti-CD47 antibody (P=0.0281). Therefore,the CD47-blocking antibody was able to produce a significant inhibitionof tumor growth.

We created a GFP-luciferase+ NCI-H82 cell line to monitor growth anddissemination in vivo. As an orthotopic model of human SCLC, weengrafted GFP-luciferase+ NCI-H82 cells into the left intrathoracicspace. Four days after injections, engraftment was confirmed bybioluminescence imaging. We then randomized mice into two cohortstreated with either vehicle control or 250 μg anti-CD47 antibody cloneHu5F9-G4 administered every other day. We monitored tumor growth overtime by bioluminescence imaging. Again, the CD47-blocking antibodyproduced a significant inhibition of tumor growth. Additionally, weobserved a significant benefit in survival for the cohort treated withthe CD47-blocking antibody. Post-mortem analysis revealed tumors formedwithin the thoracic cavity or in the parathoracic region. Mice in thevehicle control group also exhibited substantial metastases to theliver, which were not observed in the cohort treated with the anti-CD47antibody.

Since cell lines typically represent clonal populations of cells, wenext tested the in vivo efficacy of CD47-blocking antibodies on apatient-derived xenograft, which more closely models treatment inpatients since it maintains the heterogeneity of cancer cell populationswithin a tumor. Primary SCLC sample H29 was transduced to expressGFP-luciferase to allow for dynamic measurements of tumor growth invivo. Tumors were then engrafted into the lower left flanks of mice andallowed to establish for approximately 2 weeks. Mice were thenrandomized into two treatment cohorts with vehicle control or 250 μganti-CD47 antibody clone Hu5F9-G4 administered every other day. We foundthe anti-CD47 antibody significantly inhibited tumor growth, as assessedby tumor volume measurements and bioluminescence imaging (FIG. 6B-D).Treatment with the CD47-blocking therapy also produced significantbenefits in survival. By day 125 post-engraftment, all mice in thecontrol group had died whereas the majority of mice in the anti-CD47antibody group had only small tumors that failed to progress even after225 days post-engraftment (FIG. 6E). These models demonstrate thatCD47-blocking therapies could be effective for patients with SCLC.

Serum MCP-3 is a Biomarker of Response to CD47-blocking Therapies. Toidentify potential biomarkers of a response to CD47-blocking therapies,we again engrafted mice with NCI-H82 cells. We allowed tumors to grow toapproximately 1.5 cm in diameter and then we treated the mice with asingle dose of vehicle control or anti-CD47 antibody clone Hu5F9-G4. Wecollected serum samples immediately before treatment and 24 hourspost-treatment. We subjected the serum samples to multiplex analysis of38 cytokines. From this analysis, we found that macrophage chemotacticprotein 3 (MCP-3) was systemically increased following treatment withanti-CD47 antibody clone Hu5F9-G4 (FIG. 7A). No significant increase inMCP-3 was observed in mice without tumors that were treated withanti-CD47 antibody clone Hu5F9-G4 (FIG. 7A). We also performed a similarexperiment using the patient-derived xenograft H29. Again, mice bearingtumors were subjected to a single dose of anti-CD47 antibody cloneHu5F9-G4. Serum cytokine analysis again revealed that MCP-3 wassignificantly increased following treatment with the CD47-blockingantibody (FIG. 7B). Therefore, MCP-3 may serve as a biomarker ofresponse to CD47-blocking therapies in patients. Secretion of MCP-3 maybe a positive feedback mechanism that recruits more macrophages to thetumor and could in part explain the robust effects of CD47-blockingtherapies in vivo.

Comprehensive Antibody Arrays Identify Therapeutic Targets on SCLC.Monoclonal antibodies have proven to be some of the most effectivetreatments for cancer. However, there are few known antibody targets onthe surface of SCLC. For this reason, we aimed to characterize thesurface antigen profile of SCLC cells using comprehensive antibodyarrays. We subjected four SCLC cell lines and the primary SCLC sampleH29 to analysis using the BioLegend LEGENDScreen array, a comprehensivecollection of 332 antibodies to human cell surface antigens.***Discussion of histogram to define negative, low, and high antigens(FIG. 8A). We identified 39 antigens that were highly expressed on thesurface of the SCLC samples, making them possible targets of therapeuticantibodies. When we ranked these antigens by their median stainingintensity, we found that CD47 was the most intensely staining surfaceantigen (FIG. 8B). Another highly expressed antigen across all sampleswas CD56 (NCAM), a known marker of neuroendocrine tumors and atherapeutic target currently under evaluation for SCLC, thus validatingour approach. A number of other highly expressed surface antigens werealso identified that could potentially be targeted by monoclonalantibody therapies, including CD24, CD29, and CD99 (FIG. 8B).Interestingly, other immune checkpoint ligands such as CD80, CD86,PD-L1, or PD-L2 were not appreciably expressed on the surface of theSCLC samples.

Combining Antibodies with CD47-blockade Enhances Phagocytosis of SCLC.To evaluate the therapeutic potential of the antigens identified by theLEGENDScreen arrays, we next evaluated their ability to be targeted byantibodies and induce phagocytosis in vitro. We obtained antibodies to anumber of highly expressed surface antigens, including CD56 (clonesHCD56 and MEM-188), CD24, CD29, and CD99. Additionally, we obtained thesequence for lorvotuzumab, an anti-CD56 antibody being evaluated inclinical trials as an antibody-drug conjugate, and we produced itrecombinantly as a naked antibody. We tested these antibodies alone andin combination with the high-affinity CD47 antagonist CV1, which blocksCD47 but does not contribute an additional Fc stimulus (FIGS. 9A and B).We tested the ability of these antibodies to induce phagocytosis byhuman macrophages of two different SCLC cell lines, NCI-H82 (FIG. 9A)and NCI-H524 (FIG. 9B). Of the three anti-CD56 antibodies tested, wefound that lorvotuzumab was able to produce the greatest increase inphagocytosis, and this effect was significantly enhanced by combinationwith CV1. Antibodies to CD24 or CD99 were also able to inducephagocytosis that was comparable or exceeded that of treatment withanti-CD47 clone Hu5F9-G4. As expected, phagocytosis with Hu5F9-G4 wasentirely blocked when combined with CV1, since CV1 competes for the samebinding surface and binds with extremely high affinity. Interestingly,the anti-CD29 antibody was not able to induce phagocytosis even incombination with CV1, an important demonstration that additional factorssuch as surface binding geometry or the ability to engage Fc receptorsmay modify the response of macrophages to therapeutic antibodies.

Since lorvotuzumab is under evaluation as a therapeutic agent for SCLC,we investigated its ability to induce phagocytosis over a varying rangeof concentrations. Treatment with lorvotuzumab alone produced adose-response relationship for inducing macrophage phagocytosis.Importantly, we found that over each lorvotuzumab concentration tested,the addition of CV1 produced a greater degree of phagocytosis (FIG. 9C).These findings demonstrate that CV1 could increase both the maximalefficacy and the potency of lorvotuzumab, as previously observed whenCV1 was combined with rituximab, trastuzumab, and cetuximab.

Due to its poor prognosis and dearth of effective treatment options,there is an imminent need to identify novel treatments for SCLC.Immunotherapies are emerging as some of the most promising new therapiesfor cancer, and here we show that CD47, the myeloid-specific immunecheckpoint, is a genuine immunotherapeutic target for SCLC. CD47 washighly expressed on the surface of all SCLC samples tested, and we foundblocking CD47 enabled macrophage phagocytosis of SCLC samples in vitro.Using multiple xenograft models, the CD47-blocking antibody Hu5F9-G4 wasable to inhibit tumor growth and prolong survival of mice bearing SCLCtumors. Importantly, we observed anti-tumor efficacy in apatient-derived xenograft model of SCLC, which maintains the complexityof the tumor-initiating cell population and thus serves as a moreaccurate model for treatment in humans. Additionally we identified MCP-3as a serum biomarker that correlates with response to CD47-blockingtherapies. Since the anti-CD47 antibody Hu5F9-G4 is under investigationin a Phase I clinical trial for human solid malignancies(ClinicalTrials.gov identifier: NCT02216409), our findings providescientific justification for further evaluation of anti-CD47 antibodiesin subsets of patients with SCLC.

Furthermore, using comprehensive antibody arrays, we identified severalantigens on the surface of SCLC samples that could be targeted withmonoclonal antibodies therapies. Using the high-affinity SIRPα variantCV1, a next-generation CD47 antagonist, we found that CD47-blockadeaugmented the efficacy of anti-tumor antibodies for SCLC, as has beendemonstrated for other cancers. The combination of high-affinity SIRPαvariants with independent tumor-binding antibodies provided an optimalstrategy for targeting CD47 in SCLC. Blockade of CD47 on the surface ofSCLC was not sufficient to induce macrophage phagocytosis, but insteadit augmented macrophage phagocytosis when SCLC-binding antibodies arepresent. Antibodies to CD56, CD24, and CD99 proved to be effective atinducing phagocytosis of SCLC, particularly when combined with CV1.

Additionally, we found that CD47-blockade was able to enhance theefficacy of lorvotuzumab, an antibody proceeding through clinical trialsfor SCLC as an antibody-drug conjugate (ADC) with the cytotoxic agentmertansine. Combining therapeutic antibodies with CD47-blockingtherapies represents an alternative method to enhance the efficacy oftherapeutic antibodies. One benefit of CV1 over ADCs is that it can becombined with any antibody without further engineering. ADCs often relyon internalization to deliver their cytotoxic payload, and thisdependency can limit efficacy and increase side effects. Since CD47blockade stimulates macrophages to identify cells for removal, there maybe an added layer of specificity conferred by cell-cell interactionsthan that achieved by ADCs. Nonetheless, it is likely that evenlorvotuzumab-mertansine could benefit from combination with CV1 if theability to engage Fc receptors is preserved.

Our approach to identifying novel SCLC surface antigens can be appliedto other types of cancer, and in the future could be used to assembleoligoclonal cocktails of antibodies that could be used to simulate thenatural humoral immune response against foreign pathogens or cells.These cocktails could be combined with CD47-blocking therapies and otherimmunotherapies to mount an effective immune response against SCLCcells. These studies show that SCLC is responsive to CD47-blockingtherapies.

Materials and Methods

Cell lines and culture: NCI-H82, NCI-524, NCI-H69, and NCI-1688 wereobtained from ATCC. Cells were cultured in RPMI-1640 supplemented with10% fetal bovine serum (Hyclone), 1× Glutamax (Invitrogen), and 100 U/mLpenicillin and 100 ug/mL streptomycin (Invitrogen). Cell lines weregrown in suspension (NCI-H82, NCI-524, NCI-H69) and dissociated bygentle pipetting or brief incubation with 1× TrypLE (Invitrogen).NCI-1688 cells were grown in adherent monolayers and or removed by briefincubation with 1×TrypLE. Cell lines were cultured in humidifiedincubators at 37° C. with 5% carbon dioxide.

Human macrophage differentiation: Leukocyte reduction system chamberswere obtained from anonymous blood donors at the Stanford Blood Center.Monocytes were purified on an AutoMACS (Miltenyi) using CD14+ microbeadsor CD14+ whole blood microbeads (Miltenyi) according to themanufacturer's instructions. Purified CD14+ monocytes were plated on 15cm tissue culture dishes at a density of 10 million monocytes per plate.Monocytes were differentiated to macrophages by culture in IMDMsupplemented with 10% Human AB serum (Invitrogen), 1× GlutaMax(Invitrogen), and 100 U/mL penicillin and 100 ug/mL streptomycin forapproximately 7-10 days.

In vitro phagocytosis assays: In vitro phagocytosis assays wereperformed as previously described. Briefly, SCLC cancer cells wereremoved from plates and washed with serum-free IMDM. GFP-luciferase+cells or cells labeled with calcein AM (Invitrogen) were used as targetcells. Macrophages were washed twice with HBSS, then incubated with1×TrypLE for approximately 20 minutes in humidified incubators at 37° C.Macrophages were removed from plates using cell lifters (Corning), thenwashed twice with serum-free IMDM. Phagocytosis reactions were carriedout using 50,000 macrophages and 100,000 tumor cells. Cells wereco-cultured for two hours at 37° C. in the presence of antibodytherapies. After co-culture, cells were washed with autoMACS RunningBuffer (Miltenyi) and prepared for analysis by flow cytometry.Macrophages were stained using fluorophore-conjugated antibodies to CD45(BioLegend) in the presence of 100 μg/mL mouse IgG (Lampire). Dead cellswere excluded from the analysis by staining with DAPI (Sigma). Sampleswere analyzed by flow cytometry using a LSRFortessa (BD Biosciences)equipped with a high-throughput sampler. Phagocytosis was evaluated asthe percentage of calcein-AM⁺ macrophages using FlowJo v9.4.10 (TreeStar) and was normalized to the maximal response by each independentdonor where indicated. Statistical significance was determined and datawere fit to sigmoidal dose-response curves using Prism 5 (Graphpad).

Additional reagents used in phagocytosis include the high-affinity SIRPαvariant CV1 monomer, which was produced as previously described and usedat a concentration of 1 μM for blocking. Antibodies to identified SCLCantigens were used in phagocytosis assays at a concentration of 10μg/mL, including anti-CD56 (NCAM) clone HCD56 (BioLegend), anti-CD56(NCAM) clone MEM-188 (BioLegend), anti-CD24 clone ML5 (Biolegend),anti-CD29 clone TS2/16 (BioLegend), anti-CD99 clone 12E7 (Abcam).Additionally, lorvotuzumab was made recombinantly using the heavy andlight chain variable region sequences available in the KEGG database(Drug: D09927). Lorvotuzumab variable regions were cloned intopFUSE-CHIg-hG1 and pFUSE2-CLIg-hK (Invivogen) for expression.Lorvotuzumab was produced recombinantly by transient transfection of293F cells (Invitrogen) using 293fectin (Invitrogen), followed bypurification over a HiTrap Protein A column (GE Healthcare). Purifiedantibody was eluted with 100 mM citrate buffer (pH 3.0) and neutralizedwith 1/10th volume of Tris buffer (pH 8.0). Antibody was desalted usinga PD-10 column (GE Healthcare).

Sorting of macrophage populations after phagocytosis: 2.5 million humanmacrophages were combined with 5 million GFP+ NCI-H82 cells and 10 μg/mLanti-CD47 antibody (clone Hu5F9-G4) in serum-free medium and incubatedfor two hours. Macrophages were identified by staining with anti-CD45,and macrophages populations were sorted on a FACSAria II cell sorter (BDBiosciences). Cells from sorted populations were centrifuged ontomicroscope slides then stained with Modified Wright-Giemsa stain(Sigma-Aldrich) according to the manufacturer's instructions and imagedon a DM5500 B upright light microscope (Leica).

Mice: Nod.Cg-Prkdc^(scid) IL2rg^(tmWj1)/SzJ (NSG) mice were used for allin vivo experiments. Mice were engrafted with tumors at approximately6-10 weeks of age, and experiments were performed with age andsex-matched cohorts. Mice were maintained in a barrier facility underthe care of the Stanford Veterinary Services Center and handledaccording to protocols approved by the Stanford UniversityAdministrative Panel on Laboratory Animal Care.

In vivo SCLC treatment models: 1.25×10⁶ NCI-H82 cells weresubcutaneously engrafted into the flanks of NSG mice. Tumors wereallowed to grow for 8 days, then mice were randomized into treatmentgroups with PBS or 250 μg anti-CD47 antibody (clone Hu5F9-G4). Treatmentwas administered every other day by intraperitoneal injection. Tumorgrowth was monitored by tumor dimension measurements that were used tocalculate tumor volumes according to the ellipsoid formula(π/6×length×width²). For a patient-derived xenograft model of SCLC,3×10⁶ GFP-luciferase⁺ H29 cells were subcutaneously engrafted with 25%Matrigel (BD Biosciences) into the flanks of NSG mice. Tumors wereallowed to grow for 15 days, then mice were randomized into treatmentwith into treatment groups with PBS or 250 μg anti-CD47 antibody (cloneHu5F9-G4). Treatment was administered every other day by intraperitonealinjection. Tumor growth was monitored by bioluminescence imaging andtumor volume measurements as described above. Statistical significanceof tumor growth was determined by Mann-Whitney test. Survival wasanalyzed by Mantel-Cox test. Pilot in vivo experiments with H82 cellsand H29 cells were performed with smaller cohorts of mice with similarresults.

GFP-fluorescence from tumor nodules was visualized on an M205 FAfluorescent dissecting microscope (Leica) fitted with a DFC 500 camera(Leica).

Bioluminescence imaging: Mice bearing GFP-luciferase+ tumors were imagedas previously described. Briefly, anesthetized mice were injected with200 μL D-luciferin (firefly) potassium salt (Biosynth) reconstituted at16.67 mg/mL in sterile PBS. Bioluminescence imaging was performed usingan IVIS Spectrum (Caliper Life Sciences) over 20 minutes to recordmaximal radiance. Peak total flux values were assessed from theanatomical region of interest using Living Image 4.0 (Caliper LifeSciences) and were used for analysis.

Comprehensive FACS-based antibody screening: Antigens on the surface ofSCLC samples were analyzed using LEGENDScreen Human Cell Screening Kits(BioLegend), according to the manufacturer's protocol with the followingmodifications. Briefly, lyophilized antibodies were reconstituted inmolecular biology grade water and added to cell samples at a 1:8dilution. Approximately 20-40×10⁶ total cells were used for the analysisper SCLC sample. NCI-H82 was labeled with calcein-AM and analyzedsimultaneously with NCI-H524. NCI-H69 was labeled with calcein-AM andanalyzed simultaneously with NCI-H1688. The primary patient sample H69was analyzed independently. It was freshly dissociated from alow-passage xenograft and mouse lineage cells were excluded from theanalysis by staining with Pacific Blue anti-mouse H-2k^(d) (BioLegend).Samples were incubated with antibodies for 30 minutes on ice protectedfrom light. For all samples, dead cells were excluded from the analysisby staining with DAPI.

-   Jaiswal S, Jamieson C H, Pang W W, Park C Y, Chao M P, Majeti R, et    al. CD47 is upregulated on circulating hematopoietic stem cells and    leukemia cells to avoid phagocytosis. Cell. 2009; 138:271-85.-   Majeti R, Chao M P, Alizadeh A A, Pang W W, Jaiswal S, Gibbs K D,    Jr., et al. CD47 is an adverse prognostic factor and therapeutic    antibody target on human acute myeloid leukemia stem cells. Cell.    2009; 138:286-99.-   Willingham S B, Volkmer J P, Gentles A J, Sahoo D, Dalerba P, Mitra    S S, et al. The CD47-signal regulatory protein alpha (SIRPα)    interaction is a therapeutic target for human solid tumors.    Proceedings of the National Academy of Sciences of the United States    of America. 2012; 109:6662-7.-   Weiskopf K, Ring A M, Ho C C, Volkmer J P, Levin A M, Volkmer A K,    et al. Engineered SIRPalpha Variants as Immunotherapeutic Adjuvants    to Anticancer Antibodies. Science. 2013. Chao M P, Alizadeh A A,    Tang C, Myklebust J H, Varghese B, Gill S, et al. Anti-CD47 antibody    synergizes with rituximab to promote phagocytosis and eradicate    non-Hodgkin lymphoma. Cell. 142:699-713.-   Maloney D G, Grillo-Lopez A J, White C A, Bodkin D, Schilder R J,    Neidhart J A, et al. IDEC-C2B8 (Rituximab) anti-CD20 monoclonal    antibody therapy in patients with relapsed low-grade non-Hodgkin's    lymphoma. Blood. 1997; 90:2188-95.-   Vogel C L, Cobleigh M A, Tripathy D, Gutheil J C, Harris L N,    Fehrenbacher L, et al. Efficacy and safety of trastuzumab as a    single agent in first-line treatment of HER2-overexpressing    metastatic breast cancer. J Clin Oncol. 2002; 20:719-26.-   Van Cutsem E, Kohne C H, Hitre E, Zaluski J, Chang Chien C R,    Makhson A, et al. Cetuximab and chemotherapy as initial treatment    for metastatic colorectal cancer. N Engl J Med. 2009; 360:1408-17.-   Willingham S B, Volkmer J P, Gentles A J, Sahoo D, Dalerba P, Mitra    S S, et al. The CD47-signal regulatory protein alpha (SIRPa)    interaction is a therapeutic target for human solid tumors. Proc    Natl Acad Sci USA. 109:6662-7.-   Shultz L D, Lyons B L, Burzenski L M, Gott B, Chen X, Chaleff S, et    al. Human lymphoid and myeloid cell development in NOD/LtSz-scid    IL2R gamma null mice engrafted with mobilized human hemopoietic stem    cells. Journal of immunology. 2005; 174:6477-89.

1-7. (canceled)
 8. A method of treating an individual with lung cancer,the method comprising: administering to a human subject in need thereofa combination of (i) an agent that selectively blocks CD47 binding toSIRPα and (ii) a targeted therapeutic agent that specifically binds toone or more cell-surface antigens on lung cancer cells, in a doseeffective to increase depletion of the lung cancer cells.
 9. The methodof claim 8, wherein the agent that selectively blocks CD47 binding toSIRPα is an antibody.
 10. The method of claim 9, wherein the antibodyspecifically binds to CD47.
 11. The method of claim 9, wherein theantibody specifically binds to SIRPα.
 12. The method of claim 8, whereinthe agent that selectively blocks CD47 binding to SIRPα is a solubleSIRPα polypeptide.
 13. The method of claim 8, wherein the agent thatselectively blocks CD47 binding to SIRPα is a soluble CD47 polypeptide.14. The method of claim 8, wherein the one or more cell-surface antigenson lung cancer cells are selected from CD24, CD166, CD326, CD298, CD29,CD63, CD9, CD164, CD99, CD46, CD59, CD57, CD165, and EpCAM.
 15. Themethod of claim 8, wherein the combination of agents is administeredsimultaneously.
 16. The method of claim 8, wherein the combination ofagents is administered sequentially.
 17. The method of claim 8, whereinthe combination of agents is administered in overlapping dosing regimens18. The method of claim 8, wherein the individual is a human.
 19. Themethod of claim 18, wherein the lung cancer is small cell lung cancer.20. The method of claim 19, wherein said marker is selected from CD99,CD44 and EpCam.
 21. The method of claim 8, wherein the combination ofagents provides for a synergistic effect.
 22. The method of claim 10,wherein the anti-CD47 antibody comprises an IgG4 Fc region.
 23. Themethod of claim 22 wherein the antibody is 5F9-G4.
 24. A method for thetreatment of a lung cancer in a patient, the method comprising:administering to said patient an effective dose of a targetedtherapeutic agent that specifically binds to a cell surface antigenselected from CD99, CD44, EpCam, CD24, CD166, CD56, CD326, CD298, CD29,CD63, CD9, CD164, CD46, CD59, CD57, and CD165.